TLR Agonists Research Articles

Robinin inhibits pancreatic cancer cell proliferation, EMT and inflammation via regulating TLR2-PI3k-AKT signaling pathway

PurposeTo investigate the anti-tumor effect of Robinin (Toll-like receptor 2 inhibitor) in pancreatic cancer cells via regulating tumor microenvironment.MethodsThe effects of Robinin on cell proliferation or migration in Mia-PACA2 and PANC-1 were determined, using CCK8 or wound healing assay, respectively. The typical markers of EMT (αSMA and snail) and the inflammation markers (IL-6 and TNF-α) were all detected by western blot. CU-T12-9 (TLR2 agonist) was used to rescue Robinin’s effect. PI3k-p85α and Phosphorylated-AKT (p-AKT) were evaluated, compared to the β-actin and AKT, using western blot.ResultsRobinin significantly inhibited cell proliferation and migration in Mia-PACA2 and PANC-1, compared to HPNE (**P < 0.01). Robinin also attenuated the expression of α-SMA and snail in Mia-PACA2, and PANC-1 (**P < 0.01). Besides, it was found that expression of IL-6 and TNF-α were diminished in presence of Robinin in Mia-PACA2, and PANC-1 (**P < 0.01). Western blot confirmed that Robinin could target on TLR2, and further downregulated PI3k-AKT signaling pathway to exert biological function.ConclusionsRobinin exerts anti-tumor effect perhaps via downregulating inflammation and EMT in pancreatic cancer cell through inhibiting TLR2-PI3k-AKT signaling pathway. Robinin may be a novel agent in adjuvant therapy of pancreatic cancer.

Toll-like Receptor 2 Mediated Immune Regulation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

Nucleic acid sensing promotes inflammatory monocyte migration through biased coagulation factor VIIa signaling

AbstractProtease activated receptors (PARs) are cleaved by coagulation proteases and thereby connect hemostasis with innate immune responses. Signaling of the tissue factor (TF) complex with factor VIIa (FVIIa) via PAR2 stimulates extracellular signal-regulated kinase (ERK) activation and cancer cell migration, but functions of cell autonomous TF-FVIIa signaling in immune cells are unknown. Here, we show that myeloid cell expression of FVII but not of FX is crucial for inflammatory cell recruitment to the alveolar space after challenge with the double-stranded viral RNA mimic polyinosinic:polycytidylic acid [Poly(I:C)]. In line with these data, genetically modified mice completely resistant to PAR2 cleavage but not FXa-resistant PAR2–mutant mice are protected from lung inflammation. Poly(I:C)-stimulated migration of monocytes/macrophages is dependent on ERK activation and mitochondrial antiviral signaling (MAVS) but independent of toll-like receptor 3 (TLR3). Monocyte/macrophage-synthesized FVIIa cleaving PAR2 is required for integrin αMβ2-dependent migration on fibrinogen but not for integrin β1-dependent migration on fibronectin. To further dissect the downstream signaling pathway, we generated PAR2S365/T368A-mutant mice deficient in β-arrestin recruitment and ERK scaffolding. This mutation reduces cytosolic, but not nuclear ERK phosphorylation by Poly(I:C) stimulation, and prevents macrophage migration on fibrinogen but not fibronectin after stimulation with Poly(I:C) or CpG-B, a single-stranded DNA TLR9 agonist. In addition, PAR2S365/T368A-mutant mice display markedly reduced immune cell recruitment to the alveolar space after Poly(I:C) challenge. These results identify TF-FVIIa-PAR2-β-arrestin–biased signaling as a driver for lung infiltration in response to viral nucleic acids and suggest potential therapeutic interventions specifically targeting TF-VIIa signaling in thrombo-inflammation.

Inflammatory response modulation by epinephrine and norepinephrine

Relevance. Inflammation is a defense response of an organism to a pathogen. It appears in order to maintain homeostasis and is regulated by the immune, nervous, and endocrine systems. The hormones epinephrine and norepinephrine are produced in the adrenal medulla and in the brain, and are universal messengers that trigger the transmission of nerve impulses at synapses, and also have a receptor-mediated effect on immunocompetent cells. The aim of this study was to investigate adrenergic pathway regulation of inflammation on the neutrophil granulocytes in the presence of activators of innate immunity receptors. Materials and Methods. Neutrophil granulocytes were obtained from peripheral blood of healthy volunteers in a density gradient of Histopaque 1077 and Histopaque 1119 (Sigma Aldrich, Steinheim, Germany), and cultured in the presence of LPS, GMDP, epinephrine and norepinephrine. The amount of human neutrophil peptides 1-3 (HNP1-3) was examined using an enzyme-linked immunosorbent assay; the gene expression of TLR4, NOD2, ATF3 and A20 was determined using RT-PCR. Results and Discussion. Norepinephrine (noradrenaline) was found to decrease the synthesis of human neutrophils peptides 1-3 (HNP 1-3 defensins, alone and in the combination with agonists of TLR4 and NOD2 receptors - LPS and GMDP respectively. It was found out that there was no a statistically significant effect of epinephrine (adrenaline) on the production of HNP 1-3, including when combined with LPS and GMDP. As a result of the study, an increase in the levels of expression of the genes TLR4, NOD2 and regulator of inflammatory reactions A20 both in LPS- and GMDP- induced neutrophil culture were uncovered, while ATF3 was increased only in LPS-induced neutrophil culture. Epinephrine demonstrated the absence of a statistically significant effect on the expression of the studied genes. While norepinephrine significantly increased the expression of A20 genes. Conclusion. The data obtained shows that norepinephrine can reduce the synthesis of HNP 1-3, including the one induced by LPS and GMDP. Moreover, the ability of norepinephrine to induce the expression of A20 may play a significant role in modulation of inflammation.

Preliminary evaluation of safety and migration of immune activated mesenchymal stromal cells administered by subconjunctival injection for equine recurrent uveitis.

Equine recurrent uveitis (ERU), an immune mediated disease characterized by repeated episodes of intra-ocular inflammation, affects 25% of horses in the USA and is the most common cause of glaucoma, cataracts, and blindness. Mesenchymal stromal cells (MSCs) have immunomodulatory properties, which are upregulated by preconditioning with toll-like receptor agonists. The objective was to evaluate safety and migration of TLR-3 agonist polyinosinic, polycytidylic acid (pIC)-activated MSCs injected subconjunctivally in healthy horses prior to clinical application in horses with ERU. We hypothesized that activated allogeneic MSCs injected subconjunctivally would not induce ocular or systemic inflammation and would remain in the conjunctiva for >14 days. Bulbar subconjunctiva of two horses was injected with 10 × 106 pIC-activated (10 μg/mL, 2 h) GFP-labeled MSCs from one donor three times at two-week intervals. Vehicle (saline) control was injected in the contralateral conjunctiva. Horses received physical and ophthalmic exams [slit lamp biomicroscopy, rebound tonometry, fundic examination, and semiquantitative preclinical ocular toxicology scoring (SPOTS)] every 1-3 days. Systemic inflammation was assessed via CBC, fibrinogen, and serum amyloid A (SAA). Horses were euthanized 14 days following final injection. Full necropsy and histopathology were performed to examine ocular tissues and 36 systemic organs for MSC presence via IVIS Spectrum. Anti-GFP immunohistochemistry was performed on ocular tissues. No change in physical examinations was noted. Bloodwork revealed fibrinogen 100-300 mg/dL (ref 100-400) and SAA 0-25 μg/mL (ref 0-20). Ocular effects of the subjconjucntival injection were similar between MSC and control eyes on SPOTS grading system, with conjunctival hypermia, chemosis and ocular discharge noted bilaterally, which improved without intervention within 14 days. All other ocular parameters were unaffected throughout the study. Necropsy and histopathology revealed no evidence of systemic inflammation. Ocular histopathology was similar between MSC and control eyes. Fluorescent imaging analysis did not locate MSCs. Immunohistochemistry did not identify intact MSCs in the conjunctiva, but GFP-labeled cellular components were present in conjunctival phagocytic cells. Allogeneic pIC-activated conjunctival MSC injections were well tolerated. GFP-labeled tracking identified MSC components phagocytosed by immune cells subconjunctivally. This preliminary safety and tracking information is critical towards advancing immune conditioned cellular therapies to clinical trials in horses.

TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

The Defined TLR3 Agonist, Nexavant, Exhibits Anti-Cancer Efficacy and Potentiates Anti-PD-1 Antibody Therapy by Enhancing Immune Cell Infiltration.

Nexavant was reported as an alternative to the TLR3 agonist of Poly(I:C) and its derivatives. The physicochemical properties, signaling pathways, anti-cancer effects, and mechanisms of Nexavant were investigated. The distinctive characteristics of Nexavant compared to that of Poly(I:C) were demonstrated by precise quantification, enhanced thermostability, and increased resistance to RNase A. Unlike Poly(I:C), which activates TLR3, RIG-I, and MDA5, Nexavant stimulates signaling through TLR3 and RIG-I but not through MDA5. Compared to Poly(I:C), an intratumoral Nexavant treatment led to a unique immune response, immune cell infiltration, and suppression of tumor growth in various animal cancer models. Nexavant therapy outperformed anti-PD-1 antibody treatment in all the tested models and showed a synergistic effect in combinational therapy, especially in well-defined cold tumor models. The effect was similar to that of nivolumab in a humanized mouse model. Intranasal instillation of Nexavant led to the recruitment of immune cells (NK, CD4+ T, and CD8+ T) to the lungs, suppressing lung metastasis and improving animal survival. Our study highlighted Nexavant's defined nature for clinical use and unique signaling pathways and its potential as a standalone anti-cancer agent or in combination with anti-PD-1 antibodies.

Manganese@Albumin Nanocomplex and Its Assembled Nanowire Activate TLR4-Dependent Signaling Cascades of Macrophages.

The immunomodulatory effect of divalent manganese cations (Mn2+ ), such as activation of the cGAS-STING pathway or NLRP3 inflammasomes, positions them as adjuvants for cancer immunotherapy. In this study, it is found that trace Mn2+ ions, bound to bovine serum albumin (BSA) to form Mn@BSA nanocomplexes, stimulate pro-inflammatory responses in human- or murine-derived macrophages through TLR4-mediated signaling cascades. Building on this, the assembly of Mn@BSA nanocomplexes to obtain nanowire structures enables stronger and longer-lasting immunostimulation of macrophages by regulating phagocytosis. Furthermore, Mn@BSA nanocomplexes and their nanowires efficiently activate peritoneal macrophages, reprogramme tumor-associated macrophages, and inhibit the growth of melanoma tumors in vivo. They also show better biosafety for potential clinical applications compared to typical TLR4 agonists such as lipopolysaccharides. Accordingly, the findings provide insights into the mechanism of metalloalbumin complexes as potential TLR agonists that activate macrophage polarization and highlight the importance of their nanostructures in regulating macrophage-mediated innate immunity.

Evaluation of biomarker, behavioral, and immunostimulatory profiles following treatment with Class C CpG ODN in a non‐human primate model of sporadic CAA

AbstractBackgroundInnate immunomodulation has shown potential to be an effective therapeutic approach in ameliorating pathological features of Alzheimer’s Disease (AD). Our recently published study showed a reduction in cognitive deficits and AD‐related pathology in a squirrel monkey model (SQM) of sporadic cerebral amyloid angiopathy (CAA) following treatment with TLR9 agonist, Class B CpG ODN. Subsequently, we commenced a chronic treatment study evaluating the efficacy and safety of Class C CpG ODN in geriatric SQMs. Here, we further explore the efficacy of Class C CpG ODN by evaluating fluid biomarkers and implementing automated cognitive tests. Moreover, we assess the immunostimulatory profiles induced by Class B and Class C CpG ODNs using RNA‐Seq genome‐wide transcriptome analysis of SQM PBMCs.MethodGeriatric SQMs received monthly injections of CpG ODN‐C or saline (VEH) over three years. Fluid biomarkers for cerebrovascular dysfunction, neurodegeneration, and neuroinflammation were assessed by Luminex and SIMOA. Touchscreen‐based Automated Cognitive Testing System (ACTS) was applied among socially living SQMs to assess behavioral changes. RNA‐Seq analyses were performed using PBMCs isolated 24hrs following CpG ODN‐B and ‐C administrations in a separate cohort of elderly SQMs.ResultOur initial experiments confirmed the utility of SQMs for investigating plasma age‐associated neurodegeneration biomarkers, including NfL, GFAP, and neurogranin. Preliminary evaluation of cerebrovascular dysfunction‐related plasma biomarkers, such as MMPs, albumin index, bFGF, and VCAM, revealed no differences between our treatment groups at earlier time points. Although not significant, higher levels of plasma sTREM2 and YKL‐40 were observed in VEH monkeys compared to CpG ODN‐treated group. Concluding assessments will be performed in plasma collected at the end of the treatment period. Characterization of additional plasma/CSF biomarkers related to AD/neurodegeneration is underway. ACTS‐based two‐choice discrimination learning task revealed that CpG ODN‐treated group performed with a greater correct response rate than the VEH group. As expected, RNA‐Seq analyses of SQM PBMCs revealed significantly altered pathways, including Th1/Th2, TLR signaling, phagocytosis, chemokine signaling, interferon signaling, and senescence pathways, in addition to pathways involved in infiltration, activation, and recruitment of various immune cells.ConclusionThe study highlights the potential of the SQM model for future translational research on CAA/AD and for advancing innovative therapeutic interventions.

Age‐associated biomarker profiles in a non‐human primate model of sporadic CAA

AbstractBackgroundAdvancements of novel therapeutic interventions for Alzheimer’s disease (AD) have been hindered by a lack of animal models in preclinical research that naturally recapitulate the pathologic features of disease with age. Our prior work demonstrated the utility of squirrel monkeys (SQMs), a non‐human primate (NHP) model of sporadic AD pathology with abundant cerebral amyloid angiopathy (CAA), as an opportune species to assess the potential therapeutic outcomes and safety profile of our novel immunomodulatory approach using TLR9 agonist CpG‐ODN. Here we provide the first longitudinal and cross‐sectional study on aging and disease pathogenesis in SQMs by characterizing blood/CSF biomarker signatures of AD/CAA pathology.MethodSIMOA, ELISA, and Luminex platform immunoassays were utilized to investigate age‐associated profiles of plasma and CSF biomarkers involved in neurodegeneration (NfL, GFAP), brain and peripheral inflammation/glia activation (YKL‐40/CHI3L1, sTREM2), and cerebrovascular dysfunction/BBB integrity (albumin index, bFGF, VCAM‐1, MMPs).ResultCross‐sectional analysis revealed statistically significant differences in plasma NfL and GFAP levels between age groups ranging from 6 to 23 years old (NfL: one‐way ANOVA, f(6,84)) = 6.395, p&lt;0.0001; GFAP: one‐way ANOVA, f(6,84)) = 8.111, p&lt;0.0001). Preliminary assessments revealed significantly higher plasma YKL‐40/CHI3L1 and MMP1 levels, while VCAM‐1, bFGF, and MMP2 levels were significantly lower in the geriatric SQMs when compared to the young group. CSF analyses demonstrated significantly higher YKL‐40/CHI3L1 and bFGF levels, and an elevated albumin index among the geriatric cohort when compared to young monkeys. Moreover, a longitudinal follow‐up of a separate SQM cohort over three years (19‐22 years old) revealed a significant increase in plasma sTREM2 levels; neurogranin levels appeared to increase, but differences were not significant (p = 0.0625, Wilcoxon signed‐rank test). Further characterizations of fluid biomarker trajectories and their relationships with age‐associated changes in cognition and neuroimaging markers are forthcoming.ConclusionSQMs present a valuable opportunity to evaluate biomarker signatures associated with aging and the development of cerebrovascular amyloid pathology. The use of this advantageous model is important to enhance the translational potential of novel therapeutics for human benefit.

STING and TLR9 agonists synergistically enhance the immunogenicity of SARS-CoV-2 subunit vaccine

STING and TLR9 agonists synergistically enhance the immunogenicity of SARS-CoV-2 subunit vaccine

Abstract B002: Vidutolimod, an immunostimulatory virus-like particle, reduces proliferation but enhances the activation of tumor-specific T cells

Abstract One approach to enhancing the anti-tumor T cell response is to alter the tumor microenvironment (TME) through intratumoral injection (IT) of immunostimulatory agents such as Vidutolimod (Vidu). Vidu is a virus-like particle (VLP) composed of a TLR9 agonist (CpG-A, known as G10) encapsulated by the Qβ bacteriophage capsid. IT Vidu shows considerable promise in early phase clinical trials. The immune response to Vidu is initiated by induction of IFNa production by pDCs within the TME. This effect is dependent on coating of Vidu with antibodies against the Qb capsid. This is followed by a series of changes in the TME that ultimately result in an enhanced anti-tumor T cell response. Mouse models have shown that the efficacy of IT Vidu depends on the presence of both CD4+ and CD8+ T cells. Most studies to date exploring the impact of Vidu on T cells have focused on the overall T cell population. The current studies were designed to further assess the complex mechanisms by which Vidu induces an anti-tumor T cell response through use of the well-established OT-1 mouse model that allows for analysis of the tumor-specific T cell population. OT-1 mice contain CD8+ T cells with a transgenic TCR that recognizes the ovalbumin (OVA) peptide SIINFEKL sequence (OVA257-264) presented on MHC Class I. Prior to culture, OT-1 splenocytes were labeled with CellTrace Violet in order to monitor proliferation over time. OT-1 splenocytes were then cultured with EL4 cells (an OVA-negative T lymphoblast cell line) or E.G7-OVA (OVA-expressing EL4 derivative cells). Minimal proliferation or evidence of T cell activation was seen when OT-1 CD8+ T cells were cultured with EL4 cells regardless of the addition of Vidu and anti-Qβ antibodies. OT-1 CD8+ T cells cultured with E.G7-OVA cells showed both proliferation and activation as indicated by increased intracellular IFNy and surface PD-1. Addition of Vidu and anti-Qb antibody reduced OT-1 CD8+ proliferation but enhanced production of IFNγ and expression of PD-1. The increase in IFNγ and PD-1 expression was strongest in the dividing OT-1 CD8+ T cell population. Preliminary results of ongoing in vivo studies are consistent with these results. In summary, Vidu reduces proliferation but enhances phenotypic markers of activation expressed by tumor-specific CD8+ T cells (OT-1 cells) when co-cultured with cells expressing OVA, their target antigen. Markers of activation are most notable in dividing OT-1 CD8+ T cells. Citation Format: Travis D. Fischer, Caitlin Lemke-Miltner, George J. Weiner. Vidutolimod, an immunostimulatory virus-like particle, reduces proliferation but enhances the activation of tumor-specific T cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B002.

Derangements of TLR2 Likely Play a Significant Role in the Thrombosis of Ph(-) Myeloproliferative Neoplasm

Introduction . We had previously reported that TLR 2 levels were significantly elevated in patients with polycythemia vera and MPN ( PV , ET, MF grouped together ) and TLR2 levels were related to JAK2 mutation and leukocytosis ( Blood (2022) 140 (Supplement 1): 12185-12186.). Since MPN has increased incidence of thrombosis in patients with JAK2V617F mutation and leukocytosis , therefore , current studies were done to test whether TLR-2 is related to thrombosis in MPN . In vitro platelet activation ( an pre-thrombotic marker) and clinical episodes of thrombosis were then investigated. . Material and Methods. 119 patients ( 51ET, 37 PV, 31MF, 21 CTR) were studied . 1) platelet activation. : The diluted blood was incubated with platelet agonists Pam3CSK4 (TLR2 agonist) or lipopolysaccharide (LPS) (TLR4 agonist) for 15 minutes at room temperature , then platelet activation was measured by Flow cytometry assaying CD40L, P-selectin, PAC-1 and LPI ( leukocyte-platelet-interaction). We obtained more consistent results from the LPI methods . Therefore, LPI methods were used to measure the platelet activation. LPI method was measured by CD45 and CD41 antibodies (Miltenyi Biotec, Auburn, CA), and platelet activation were measured as the percentage of CD45 cells with aggregated CD41. To test whether TLR2 or TLR4 were responsible for the LPI response ,TLR2 inhibitor C29 (InvivoGen, San Diego, CA) or the TLR4 inhibitor C34 ((Sigma-Aldrich, St. Louis, MO) were added to the LPI assay. Effects of inhibitions were measured by fold-changes which were calculated by dividing the LPI values after activation by either Pam3CSK4 (TLR2 agonist) or LPS (TLR4 agonist), with or without added inhibitors, by the LPI values before activation or inhibition. Results . 1) As shown in Fig A, a) un-activated MPN (U-MPN) patients had more LPI than un-activated controls (u-CTR), with mean ± SE of 13.01 ± 1.12 and 5.01 ± 0.97 in U-MPN and U-CTR, respectively (P&amp;lt;0.01). This is consistent with previous reports that un-activated MPN patients have more intrinsically activated platelets than un-activated controls .b) Greater LPI response by activation by Pam3CSK4, (TLR2 agonist ) than by LPS ( lipopolysaccharide)) (TLR4 agonist) . MPN and controls ( mean ± SE ) were of 22.25 ± 0.99 and 10.46 ± 0.44 ( by Pam3CSK4) and 12.12 ± 2.16 and 4.34 ± 0.32 ( by LPS) (P&amp;lt;0.0005) respectively . 2). Fig B showed the TLR2 inhibitor C29 significantly suppressed LPI ( measured by fold-changes ) , in un-activated MPN (0.38 ± 0.05), activated controls (0.42 ± 0.11), and activated MPN (0.42 ± 0.05). Conversely, the TLR4 inhibitor C34 had no inhibitory effect on un-activated MPN (0.98 ± 0.03), activated MPN (1.02 ± 0.02), or activated controls (0.84 ± 0.06) . 3) As shown in Table 2 , TLR 2 levels elevated Patients ( TLR2-E) ( defined as more than mean plus 2 standard deviation of controls (&amp;gt; 310 MFI) , have more thrombosis episode (29%) than TLR2 -normal patients (11%) . Conclusion . TLR2 likely plays a significant role in thrombosis in MPN patients on the basis : 1) JAK2 mutated patients have more TLR2 levels 2) TLR2-E patients have more leukocytosis than TLR-N patients 3) LPI was significantly elevated with TLR2 agonist ( Pam3SK4) but not by TLR4 agonist ( LPS) , and LPI was significantly decreased by TLR2 inhibitor and not by TLR4 inhibitor. 4) clinical evidence of more thrombosis in TLR -E patients than TLR-N patients.

Interferons Prime Endothelium for TLR-Mediated Prothrombotic Transformation

Cytokine storm occurs with respiratory infection and is associated with microvascular thrombosis. However, the cytokines responsible for promoting thrombus formation in the pulmonary microvasculature and the mechanisms by which they act are largely unknown. In its initial response to invading viruses or bacteria, pulmonary epithelium and macrophages release a variety of cytokines into the pulmonary circulation. With progressive invasion, the fragile barrier between alveolus and pulmonary capillaries breaks down and endothelial cells are exposed to pathogen-associated molecular patterns (PAMPs), which stimulate endothelial toll-like receptors (TLRs). To identify the cytokines that stimulate endothelial prothrombotic activity, we screened MIP-1β, MCP-1, IL-10, IL-6, IL-1β, TNFα, IFNα, IFNβ, and IFNγ for their ability to synergize with PAMPs in order to stimulate thrombin generation on endothelium. Dose curves showed that IFNs and TNFα were ~1000-fold more potent than the other cytokines at priming endothelium for thrombin generation induced by poly(I:C), a synthetic PAMP that stimulates TLR3 ( Fig. 1). Anti-tissue factor (TF) antibody completely attenuated thrombin generation induced by either IFNα or IFNγ exposure followed by poly(I:C) stimulation. Evaluation of TF expression and FXa generation showed that although exposure of endothelium to IFNs did not itself enhance TF expression or activity, IFNs substantially augmented TF expression and activity in response to poly(I:C). In addition, IFNα and IFNγ significantly inhibited thrombomodulin expression, even in the absence of poly(I:C). To evaluate whether or not IFNs enhance thrombin generation in endothelium stimulated through TLRs other than TLR3, we exposed endothelium to IFNα or IFNγ, followed by stimulation with different TLR-selective agonists. IFNγ exposure enhanced thrombin generation on endothelium in response to the TLR1/2 agonist PAM3CSK4, but IFNα did not have a significant effect, indicating the specificity of coupling to a particular IFN subtype. In contrast, the stimulation of endothelial thrombin generation by the TLR3 agonists, poly(I:C) and poly(A:U), was more potently enhanced by IFNα compared to IFNγ. Stimulation of thrombin generation by TLR4 agonists, including LPS and CRX-527, was significantly enhanced by IFNγ, but not IFNα. This coupling of IFNα with TLR3 agonists is consistent with the known association of viral infection with IFNα stimulation and TLR3 activation. Coupling of IFNγ with TLR2/4 agonists reflects a previously identified association of bacterial infection with both IFNγ stimulation and TLR 2 and 4 activation. Evaluation of conditioned media from pulmonary epithelial cells showed that media from inflamed pulmonary epithelial cells, but not quiescent epithelium, primed endothelium for substantial thrombin generation in response to poly(I:C). Anti-IFNα antibodies blocked the priming effect of inflamed condition media. These studies show that endogenous IFNs elaborated by pulmonary epithelium prime endothelium for TLR-mediated thrombin generation. These results show that IFNs are both necessary and sufficient to enhance TLR-mediated endothelial thrombin generation and provide a new framework for understanding the role of IFNs in cytokine-related thrombus formation ( Fig. 2). This model indicates that endothelial cells possess a verification system wherein a robust thrombotic response is only initiated if the type of IFN released by alveolar macrophages and epithelium is consistent with the type of TLR sensed by the endothelium. These observations provide a mechanistic basis for microvascular thrombosis in cytokine storm and raise the possibility of targeting interferons to prevent thrombotic complications in this setting.

Mitochondrial Damage-Associated Molecular Patterns (DAMPs) Contribute to the Development of Murine Antibody-Mediated Transfusion-Related Acute Lung Injury (TRALI)

Transfusion-related acute lung injury (TRALI) is potentially life-threatening and has been ranked as the leading cause of transfusion-related fatalities since its description in the 1980s. With a 5 to 14% mortality rate, symptoms range from mild dyspnea, cough and fever to full-blown respiratory failure and severe hypoxemia, which usually occurs within 6 hours from the transfusion. To date, the pathogenesis is not fully understood, but it appears that TRALI requires two hits in order to be initiated; the first-hit is the patient's clinical condition often reflected by a state of inflammation while the second hit is the transfusion itself. Several published articles have shown that mitochondrial damage-associated molecular patterns (mtDAMPs) including extracellular mitochondria and mitochondrial DNA (mtDNA) are elevated in stored transfusion products that have been linked to adverse transfusion reactions. Using an in vivo murine model of TRALI where injection of a monoclonal anti-MHC class I antibody (34-1-2s) into LPS-primed BALB/c mice causes significant lung edema, we explored the possibility of whether mitochondria and mtDAMPs could act as first hits in TRALI induction. Mitochondria were prepared from mouse livers by differential centrifugation and purified on a density gradient. Intravenous injections of only mitochondria (2.5 x 10 8 per mouse) prior to injection with 34-1-2s caused TRALI characterized by significantly elevated lung wet/dry ratios (Figure 1A), temperature loss and lung neutrophil accumulation. In contrast, however, intraperitoneal injections of mitochondria did not prime as a first hit for 34-1-2s-induced TRALI suggesting that the route of the mitochondrial first hit is important in determining whether TRALI proceeds (Figure 1A.). Using a TLR9 agonist (ODN2395) to mimic mtDNA as a first hit, we also observed 34-1-2s-induced TRALI induction, and this could be prevented by the administration of a specific TLR9 antagonist (ODN4084F) 2h before the ODN2395 injection (Figure 1B). Of interest, the TLR9 antagonist was not sufficient to prevent TRALI induced by the iv whole mitochondria first hit, indicating that other mtDAMPS are also involved in the priming of the mice for TRALI. This study suggests that mitochondria and at least mtDNA can act as a first hit in antibody-mediated TRALI via stimulation of recipient TLR9 and that the first hit route maybe important in stimulating TRALI. Further investigations are underway to identify other mtDAMPS (such as N-formyl peptides) or a combination of DAMPs that are important for the first hit. Inhibition of mitochondrial DAMPS may be a potential therapy for transfusion recipients suffering from TRALI.

An Idiotype Vaccine Strategy Utilizing Targeted NGS and Cell Free Protein Synthesis System Ensures Identification and Production of Human Idiotype Antigens, and Shows Promising Results in Preclinical Models of B-Cell Lymphoma with a Novel TLR7/8 Agonist-Based Adjuvant and Anti-PD-L1 Antibody

Background: Non-Hodgkin lymphoma (NHL) is one of the most common hematological malignancies in the world. Despite advances in treatment options, such as chimeric antigen receptor T cells (CAR-T) or bispecific antibodies, many patients still recur after complete remissions (CR) or do not achieve CR. Since B cell malignancies arise from clonal expansion, the B cell receptor (BCR) expressed on the cell surface, idiotype, has been studied as a tumor-specific antigen. Several trials have evaluated idiotype vaccines in clinical setting, but failed to demonstrate efficacy. Limitations have arisen from the uncertainty of BCR sequence confirmation, long turnaround time in manufacturing idiotype antigen, and the absence of strong immune modulators. Here, we utilized targeted next generation sequencing (NGS) for BCR, cell-free protein synthesis (CFPS) system for idiotype antigen manufacturing, and a novel liposomal TLR7/8 agonist-based adjuvant to optimize idiotype vaccine. Methods: To generate the idiotype antigens as single-chain variable fragment (scFv) from A20 lymphoma cell line and biopsy samples from NHL patients, variable regions of BCR heavy and light chain were sequenced by targeted NGS. Total RNA was isolated from A20 cells and tumor tissues from patients, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using constant region-specific primers, resulting in the 1 st strand cDNA. Subsequently, the 2 nd strand cDNA was synthesized using V-gene specific primers and sequenced using an Illumina MiSeq sequencer. We applied targeted NGS to patients' tumor tissues from Asan Medical Center and Seoul National University Hospital to understand whether this method works in human tissues as well. The identified sequence was cloned into a linear DNA template and expressed by CFPS system. We optimized the CFPS system and purification process of idiotype antigen scFv from A20 cell line and human tissues. The liposomal adjuvant ProLNG-001 (TLR7/8 agonist 140 μg) and ProLNG-002 (TLR7/8 agonist 70 μg + TLR4 agonist 5 ug) were synthesized by microfluidics method. Idiotype antigen A20-scFv 50 μg was given alone or mixed with ProLNG-001 or -002. To investigate the efficacy of the idiotype vaccine, BALB/c mice were subcutaneously injected with 5×10 5 of A20 cells. Mice were subcutaneously vaccinated 4 times at 1-week intervals starting from day 2 after tumor inoculation. Atezolizumab 10 mg/kg was administered intraperitoneally 3 times a week for 3 weeks in the idiotype vaccine + immune checkpoint inhibitor combination group (Figure 1a). Results: Targeted NGS has confirmed BCR sequences from A20 cell line and dominant malignant B cell clone from patients' tumor with a success rate of over 90% when sufficient volume (more than 500 mm 3) of biopsy sample was provided for RNA extraction. Also, we were able to express the idiotype scFv antigens determined from 18 NHL patients' tumor samples using CFPS system with a 100% success rate (Figure 1b). The untreated mice (Group 1) showed a tumor growth pattern similar to that of Group 2 (vaccination with antigen only). Vaccination with idiotype antigen mixed with liposomal adjuvants, ProLNG-001 (Group 3) and ProLNG-002 (Group 4), significantly inhibited tumor growth. The combination of anti-PD-L1 antibody and idiotype antigen mixed with ProLNG-002 (Group 5) generated even better overall anti-tumor responses with 14 out of 15 mice showing no residual tumor at day 34 after tumor inoculation. Conclusion: Taken together, the idiotype vaccine strategy using targeted NGS for BCR, CFPS system for idiotype antigen manufacturing, and TLR7/8-agonist-based liposomal adjuvants showed highly effective results in the preclinical syngeneic A20 tumor model. Furthermore, the usage of immune checkpoint inhibitors combined with idiotype vaccine in B cell lymphoma may exert synergistic effects. The CFPS system can be applied to swift, robust and personalized manufacturing of idiotype antigen for individuals with different idiotype sequence in a GMP setting, which was not possible in prior studies. These results support further development of the idiotypic vaccine as a treatment option for non-Hodgkin's B cell lymphomas.

Inflammatory Response to Intravenously Administered Lipopolysaccharide (LPS) in the Bone Marrow of Healthy Volunteers As a Model for Early Phase Drug Development

Introduction Inflammatory responses in the peripheral and central hematological compartments are important for identification of new biomarkers and drug targets in early phase drug development. There is an increasing interest to investigate the modulation of the inflammatory response upon experimental drug exposure using cellular and molecular analyses, specifically in the central compartment. The lipopolysaccharide (LPS) challenge model allows to study TLR4-mediated local and systemic inflammation and therefore the effects of anti-inflammatory compounds. In addition, there is an interest in the effects of TLR4 agonists on early immune cell populations. The aim of the current study is to investigate the effects of intravenous (i.v.) LPS on the peripheral blood and central bone marrow compartment and to perform a proof-of-methodology study for bone marrow analysis in early phase drug research in healthy volunteers. Methods This was an open-label study in 13 healthy young male subjects, aged 18 - 35 years (inclusive). The study was conducted at the Centre for Human Drug Research, Leiden, The Netherlands. The study protocol was approved by the Medical Ethics Committee. All subjects provided written informed consent prior to any study activity. Subjects were divided into control group (n=3), 1 ng/kg i.v. LPS group (n=5) and 2 ng/kg i.v. LPS group (n=5). Blood and bone marrow samples were collected at baseline and at 4h post-dose timepoints. Primary objective was to evaluate the safety and tolerability of bone marrow sampling in healthy volunteers, by continuous safety monitoring, visual analogue scale (VAS) for pain and subject experience questionnaires. To investigate the pharmacodynamic (PD) effects of i.v. LPS challenge on the bone marrow and whole-blood immune compartments, flow cytometry, cytokine analysis and RNA sequencing was performed. Results All 13 subjects completed the study. Adverse events were mild and reversible, and included puncture site pain related to bone marrow puncture, and flu-like symptoms related to LPS administration. Twelve subjects (92%) indicated they would undergo the procedure again in the future. There was an observable difference in VAS pain score between the baseline bone marrow puncture and LPS treatment groups, but no difference between 1 ng/kg and 2 ng/kg groups. Phenotypic changes of inflammatory signatures in peripheral blood and bone marrow cell populations upon LPS administration were detected by multi-parameter flow cytometry. An LPS-induced inflammatory cytokine profile was present. RNA sequencing revealed differential gene expression in inflammatory gene signatures between peripheral blood mononuclear cells and bone marrow cells. Conclusion We demonstrated that bone marrow analysis is feasible and well-tolerable in early phase studies with healthy volunteers and can be used to assess inflammatory responses in the central hematological compartment upon systemic LPS challenge. Access to bone marrow can provide important novel insights into drug effects on the hematopoiesis and differentiation of immune cells and provides added value to early drug development.

Designing a multi-epitope vaccine against Peptostreptococcus anaerobius based on an immunoinformatics approach

Peptostreptococcus anaerobius is an anaerobic bacterium, which has been found selectively en-riched in the fecal and mucosal microbiota of colorectal cancer (CRC) patients. Emerging evidence suggest P. anaerobius may contribute to the development of CRC in human. In this study, we designed a multi-epitope chimeric vaccine against P. anaerobius PCWBR2, a recently identified adhesin that interacts directly with colon cell lines by binding α2/β1 integrin frequently overexpressed in human CRC tumors and cell lines. Immunoinformatics tools predicted six cytotoxic T lymphocyte epitopes, five helper T lymphocyte epitopes, and six linear B lymphocyte epitopes. The predicted epitopes were joined with AAY or GPGPG linkers and a previously reported TLR4 agonist was added to the vaccine construct's N terminal as an adjuvant using EAAAK linkers and the order of epitopes was optimized. Further in silico analysis revealed that the vaccine construct possesses satisfactory antigenicity, allergenicity, solubility, physicochemical properties, adjuvant-TLR4 molecular docking, and immune profile characteristics. Our study provided a promising design for vaccines against P. anaerobius.

Poly I:C Pre-Treatment Induced the Anti-Viral Interferon Response in Airway Epithelial Cells.

Type I and III interferons are among the most important antiviral mediators. Increased susceptibility to infections has been described as being associated with impaired interferon response in asthmatic patients. In this work, we focused on the modulation of interferon dysfunction after the rhinovirus infection of airway epithelial cells. Therefore, we tested polyinosinic:polycytidylic acid (poly I:C), a TLR3 agonist, as a possible preventive pre-treatment to improve this anti-viral response. In our human study on asthma, we found a deficiency in interferon levels in the nasal epithelial cells (NEC) from asthmatics at homeostatic level and after RV infection, which might contribute to frequent airway infection seen in asthmatic patients compared to healthy controls. Finally, pre-treatment with the immunomodulatory substance poly I:C before RV infection restored IFN responses in airway epithelial cells. Altogether, we consider poly I:C pre-treatment as a promising strategy for the induction of interferon response prior to viral infections. These results might help to improve current therapeutic strategies for allergic asthma exacerbations.

Poly(I:C)/c-GAMP/Alum ternary adjuvant system synergistically enhances the immunogenicity of glycopeptide antigen

The highly glycosylated mucin 1 (MUC1) serves as a promising target for cancer vaccines. However, the weak immunogenicity of MUC1 greatly impedes its application. Here, we covalently linked a MUC1 glycopeptide to carrier protein bovine serum albumin (BSA), and the resulting BSA-MUC1 conjugate was integrated with a ternary adjuvant system comprising TLR3 agonist poly(I:C), STING agonist c-GAMP, and Alum adjuvant. Compared to adjuvant-free and Alum-adjuvanted groups, the ternary adjuvant group induced 18.3- and 10.9-fold increases in MUC1-specific IgG titers, and effectively suppressed tumor growth. These findings may propose a straightforward yet highly effective strategy for designing vaccines, offering promising prospects in cancer treatment.